Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.     

Release of a potent polymorphonuclear leukocyte chemoattractant is regulated by white-opaque switching in Candida albicans

Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.     

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into ‘neighborhoods’ comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.     

Association of DLG5 R30Q variant with inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal system known as the inflammatory bowel diseases (IBD). Recently, Stoll and colleagues reported a novel finding of genetic variation in the DLG5 gene that is associated with IBD (CD and UC combined). We present here a study of the genetic variation described in that report in two well-powered, independent case-control cohorts and one family-based collection, and confirm the proposed association between IBD and the R30Q variant of DLG5 in two of the three studies. We are, however, unable to replicate the other proposed association to the common haplotype described in Stoll et al and suggest that this other finding could conceivably have been partially a statistical fluctuation and partially a result of LD with the replicated R30Q association. This study provides support for the hypothesis that DLG5 constitutes a true IBD risk factor of modest effect.     

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.     

A mouse model of hepatocellular carcinoma: ectopic expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice

Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic alpha-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2'-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for beta-catenin. Sequencing of the tumor DNA encoding beta-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

An investigation into the relationship between salivary cortisol,stress, anxiety and depression

This study examined the relationship between indices of self-reported emotional distress and absolute versus change in cortisol levels. Fifty-four women attending a diagnostic breast clinic completed scales measuring stress, anxiety and depression and provided five saliva samples over the course of a single day for the measurement of cortisol. No significant relationships were evident between absolute cortisol levels and the distress measures. Analysis of the change in cortisol levels revealed a non-linear interaction effect between stress and anxiety and time of day. There was a non-linear relation between time of day and cortisol levels, but the extent of the non-linearity was dependent upon levels of stress and anxiety, not depression. A relationship was apparent between indices of distress and change in cortisol levels, but not absolute levels of the hormone.